[RASMB] Bad scans from XLA

John Philo jphilo at mailway.com
Thu Oct 2 13:35:09 PDT 2008


James, after seeing the 2nd, correct PDF I notice one other symptom: in that
last experiment the data all the way at the left in the air-air space are
actually changing with time, which obviously should never happen, and seem
to be extraordinarily noisy too. So that does suggest there may be a problem
with the optics or slit assembly movement.
 
Sorry, if I'd paid attention to the sedimentation coefficient scale it
should have been obvious you are running at low speeds so I strongly doubt
the leaks caused bending of the centerpiece rib.
 
John

  _____  

From: James [mailto:james.falabella at nist.gov] 
Sent: Thursday, October 02, 2008 12:58 PM
To: jphilo at mailway.com; rasmb at server1.bbri.org
Subject: RE: [RASMB] Bad scans from XLA



John,

 

Thank you for your insight into the problem, I will make certain that the
reference meniscus does not move throughout the run. I am using 15,000 rpm
for the 10 nm gold particles and 5,000 rpm for the 20 nm particles I will
apply the other corrections and hopefully the data will not show a bent
center rib. I will run another radial calibration at 3000 rpm to ensure that
this is not the issue. I will also check the masks in the centerpiece to
ensure that they are not inserted upside down. I think also my fit of the
data may be a problem too with the right boundary too far over and I will
correct this in the future data analysis. 

 

If all of this does not work, I will call Beckman to clean the slit assembly
since I am a rather new user with about a year of experience using the XLA.

 

Thank you so much for your advice on rectifying this problem that I am
experiencing. I will follow up with results when I try these things.

 

James

 

  _____  

From: John Philo [mailto:jphilo at mailway.com] 
Sent: Thursday, October 02, 2008 3:27 PM
To: 'James'; rasmb at server1.bbri.org
Subject: RE: [RASMB] Bad scans from XLA

 

James,

 

I am wondering if you are possibly getting intermittent leakage between the
channels as the rotor accelerates. This can sometimes make it seem like
sedimentation has started somewhere far away from the true meniscus
position, as seems to be the case in the second 'bad' run. In that data your
reference meniscus seems to be at ~5.96 cm, i.e. not right on top of the
sample meniscus as it will be if there is extensive cross-channel leaking,
but possibly the reference started out significantly further to the left and
moved as the rotor accelerated.

 

You also want to be very careful about aligning the cell in the rotor or you
will get convection (and very screwy data).

 

Another possibility is that your leaked gold particles are getting into the
slit assembly and causing problems with the radial scanning. 

 

I'm not clear what rotor speed you are using but when you leak out half a
sample and the speed is high enough the center rib of the centerpiece can
get permanently bent, after which you will always get strange data with that
centerpiece.

 

I am not clear why you keep doing radial calibration and in general I would
not recommend this, but I doubt that is the fundamental problem here. The
calibration should not change during normal operation---it will only change
if someone works on the optical components. Also you should not turn off the
delay calibration. Definitely never ask for radial calibration at speeds
above 3000 rpm.

 

Having said that I do wonder whether your radial calibration is correct. It
looks to me like you are getting good data (flat plateaus) well beyond 7.14
cm, which is typically not true for the standard epon centerpieces and
window holders, which suggests the radial calibration may be faulty. The
true base of the channel should be very close to 7.2 cm (and seems not to be
positioned correctly in your fits). If the radial cal is bad it might be the
counterbalance itself. The masks that slip into the sides of the
counterbalance to create the precision reference points can fall out and
hence can be put back in upside down.

 

John

 

  _____  

From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] On
Behalf Of James
Sent: Thursday, October 02, 2008 10:38 AM
To: rasmb at server1.bbri.org
Subject: [RASMB] Bad scans from XLA

Dear RASMB members,

 

I have recently been struggling with a recurring problem when I centrifuge
10 and 20nm gold particles that causes my XLA to generate poorly formed OD
data scans that cannot be fit by Sedfit. I had initially been getting well
formed scans that were easily fit by Sedfit using the continuous c(s) model
however after half of the sample side in one cell (in a 4 cell rotor) leaked
out during a run I was no longer able to get the nice sigmoidal scans that I
originally had even though I tightened and refilled the leaking cell. I
asked the Beckman service rep to investigate and he cleaned the optics and
told me to have the XLA perform a radial calibration and the scans were back
to a sigmoidal shape using the same sample of gold nanoparticles and Sedfit
could fit the data well again. However after another cell leaked in the
centrifuge, the scans could not be fit with Sedfit even though the cell was
tightened and refilled and no longer leaked. I have been performing a radial
calibration before each run and have not turned off the delay calibration
hoping the instrument will recalibrate and improve the scans however, this
has not happened. I have attached screen shots showing the quality of the
data before and after the cell leak. It looks like it could be the optics
but I am not convinced this is the whole story since it seems extreme that
one cell leak can alter the XLA performance this much until the optics are
cleaned.

 

 

James B. Falabella

Process Sensing Group

National Institute of Standards and Technology Gaithersburg, MD

Tel: 301-975-5041

Fax: 301-975-2643

 

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